Our application addresses broad Challenge Area 11, "Regenerative Medicine", and specific Challenge Topic 11-GM-101, "Establishment of regenerative capabilities". In adult mammals, muscle stem cells (MuSCs) proliferate less, there is less new muscle produced, and there is more fibrosis, than in young mammals. These effects are at least partly due to soluble molecules that regulate MuSCs and their progeny (Conboy, 2005). The concentrations of these molecular regulators exhibit temporal variation during regeneration, and this time-dependence is important for successful MuSC activation and preventing fibrosis (Brack, 2007). Our goal here is to deliver these regulators with appropriate temporal profiles to establish regenerative capabilities of MuSCs and their progeny in adult mice. Our proposal addresses the fact that there is currently no technology to vary the concentrations and timing of regulator molecules in a physiologic manner in mice without either genetic engineering, which is difficult to do with more than one regulator simultaneously and also requires breeding for generations and is thus slow, or by mechanically connecting the mouse to a pump via an external tether, which has several disadvantages: tethers prevent group housing and are incompatible with many behavioral and imaging assays performed on mice. To meet this challenge we invented a mouse-implantable remote-control micropump. We have successfully used the pump to deliver luciferin to mice carrying MuSCs expressing the firefly enzyme luciferase, causing the MuSCs to emit light (bioluminescence) in a dose-dependent and time-dependent manner. We implant the pump under the skin of the back and run a catheter under the skin and under the fascia of the muscle carrying the luciferase-expressing MuSCs. We hypothesize that our pump can deliver stem cell regulator molecules to the MuSCs on a long-term basis during regeneration using physiologic temporal dosage profiles. These profiles will be automatically synchronized with mouse behaviors including exercise and sleep in the case of candidate MuSC regulators that are naturally synchronized with these behaviors, such as IGF-1. To this end we have built custom cages with sensors in running wheels and cameras which track infrared light emitters in the implanted pumps, allowing us to determine automatically which mouse is exercising at any given time and send a radio signal to the pump in that mouse to instruct the pump to deliver the MuSC regulator. Our pump is currently made by hand, a long and tedious process. Our specific objectives are to translate our current working pump prototype into a mass-producible version that two local companies can manufacture in sufficient quantities to achieve statistical significance (also supporting local employment), to test our pumps and custom cages using a regulator of regeneration with a known temporal profile (IGF-1), and then to identify effective temporal profiles for 5 other soluble regulators of MuSCs and their progeny (Wnt7a, testosterone, HGF, Wnt3a inhibitor, and MGF). Our model will be the same as for our luciferin pump test: non-luciferase mice transplanted with luciferase-expressing MuSCs. After the MuSCs have had time to engraft we will injure the muscle using a snake venom myotoxin, notexin, an injury model with which we have experience. We will use old mice and young controls, because the regenerative deficits we aim to ameliorate are easier to measure in old mice. Specifically, our readouts will be: 1) bioluminescence to quantify the proliferative response;2) histology to quantify fibrosis, apoptosis, and the phenotypes of the cells producing the bioluminescence signal;and, 3) functional assays to measure animal gait and mobility and muscle contractile force. We will try two temporal profiles for each regulator. The first two temporal profiles will bracket a range estimated based on the literature. The third temporal profile will be further informed by our experience with the first two temporal profiles. Given the strong interest in establishing regenerative capabilities in adult cells in situ to improve wound healing and reduce scarring, a means of stimulating stem cell function in situ using physiologic temporal dosage profiles should be attractive, provide a valuable tool for the regenerative medicine field, and ultimately impact regeneration of tissues in humans. Economic impact: Our proposal would positively impact the economy by directly creating or retaining 8 jobs at Stanford Medicine, Stanford Engineering, and at local companies EoPlex (Mountain View, CA) and BesTek (San Jose, CA) (see supporting letters). In addition, according to the California Biomedical Industry 2009 report, for every individual directly employed by Stanford Medicine there is a multiplier effect, with another three to five people employed in firms that offer goods and services. Successful tissue regeneration in adults will likely require multiple drugs delivered to specific tissues with specific temporal (time-varying) dosage patterns. Currently there is no way to deliver time-varying patterns of multiple drugs to mice in a manner compatible with standard group housing and standard behavioral and imaging assays, significantly delaying the arrival of regenerative medicine therapies for humans. Our mouse- implantable remote-control micropump technology overcomes this limitation;enabling more rapid, efficient, and economical discovery and delivery of regenerative therapies.